Exploring the Gas‐Phase Formation and Chemical Reactivity of Highly Reduced M8L6 Coordination Cages

Abstract Coordination cages with well‐defined cavities show great promise in the field of catalysis on account of their unique combination of molecular confinement effects and transition‐metal redox chemistry. Here, three coordination cages are reduced from their native 16+ oxidation state to the 2+ state in the gas phase without observable structural degradation. Using this method, the reaction rate constants for each reduction step were determined, with no noticeable differences arising following either the incorporation of a C60‐fullerene guest or alteration of the cage chemical structure. The reactivity of highly reduced cage species toward molecular oxygen is “switched‐on” after a threshold number of reduction steps, which is influenced by guest molecules and the structure of cage components. These new experimental approaches provide a unique window to explore the chemistry of highly‐reduced cage species that can be modulated by altering their structures and encapsulated guest species.


General Materials and Considerations
Tetrakis(4-aminophenyl)porphyrin was obtained from Combiblocks and 2-pyridinecarboxaldehyde and C 60 -fullurene from Merck Sigma-Aldrich and used as received. Tetrakis(4-aminophenyl)porphyrin nickel(II) and cages Ni-1, C 60 ⊂Ni-1 and H 2 -1 were prepared according to literature procedures. [1] Solution nuclear magnetic resonance (NMR) spectra were recorded on either a Bruker Avance 600 or 400 MHz spectrometer and referenced to the relevant solvent peak. When mixtures of DMF-d 7 and acetonitrile-d 3 were used, spectra were referenced to the residual solvent peak of the latter.

Electron Transfer Mass Spectrometry
All experiments were performed with a high-resolution Orbitrap Elite mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) operating at a mass resolution of 60,000 (at m/z 400). Mass calibration in positive ion mode was performed prior to use. Target compounds were dissolved in acetonitrile at approximately 0.1 mg/mL, then diluted further into acetonitrile to achieve a final concentration of ca. 0.5 μM. Diluted samples were infused into the heated electrospray ionisation (HESI) source at 15 μL/min. Typical ion source parameters were source voltage 4.2 kV, capillary temperature 275 °C, sheath gas 25 arbitrary units, auxiliary gas 2 arbitrary units, S-lens RF level 53%. Mass-to-charge ratios of precursor ions and reaction products were within 5 ppm of their predicted values (Tables S4-9), using the most abundant peak in the isotopic distribution (base peak) for comparison. Ion-ion reactions of cage cations with fluoranthene radical anions (FL -• ) were performed using the electron transfer dissociation (ETD) module of the Orbitrap Elite, [2] with a reagent vial temperature of 108 °C and an automatic gain control (AGC) target set to 4 × 10 5 . Multiply charged analyte cations (from ESI) were mass-selected in the ion trap with the smallest isolation window allowing maximum retention of ions of interest (2.5 -15 Da) and the ion trap stability parameter (q) set to 0.25. Fluoranthene ions were generated by chemical ionisation and transferred into the ion trap for a given reaction time before the cationic reaction products were scanned out and analysed in the Orbitrap.

Ion Mobility Mass Spectrometry
Travelling wave ion mobility mass spectrometry (TWIM-MS) uses a travelling electric field to transmit ions through a buffer gas. [3] The time taken for ions to complete this transit is known as drift time.
Following instrument calibration using known standards, drift time can be used to determine an ion's collision cross section (CCS), which is related to the ion's size and shape. Changes in CCS occurring over the course of a series of chemical reactions can inform on the presence of resulting conformational rearrangements. [4] TWIM-MS experiments of gas phase electrochemically reduced ions were performed on a Waters Synapt G2-Si mass spectrometer in "ETD" mode with 1,4-dicyanobenzene radical anions (m/z 127) generated from a glow-discharge source as the gas phase electron donor. The spray capillary voltage, sampling cone and source offset were set to 2.5 kV, 0 V and 0 V respectively. The trap entrance, bias, trap DC and exit were set at −3, 30, −3 and 10 V. Mobility separation experiments were carried out on electrochemically reduced ions that had been quadrupole mass-selected using settings adapted from a previous report [4] with a travelling wave velocity of 1500 ms −1 and wave height of 40 V in the ion mobility cell, and 220 ms -1 and 4 V in the transfer cell. Trap and transfer cells were filled with Ar at 2.4 × 10 −2 and 2.8 × 10 −2 mbar respectively, the helium cell with He at 3.81 mbar and the ion mobility with N 2 at a pressure of 2.98 mbar. Radical anions were generated with a glow discharge current of 55 µA and a make-up gas flow of 30 mLmin −1 . The ion mobility cell was calibrated for N 2 collisional cross section (CCS) determination according to a previously described method [5] using the arrival times of cytochrome C (equine) and Myoglobin (equine) dissolved in 50:50 methanol:water. Drift tube IM-MS nitrogen buffer gas CCS reference values were obtained from a previous report. [6] In individual experiments, mass-selected ions of Ni-1(OTf) 5 11+ , Ni-1(OTf) 7

9+
, C 60 ⊂Ni-1(OTf) 5 11+ and C 60 ⊂Ni-1(OTf) 7 9+ were subjected to gas phase radical ions and the resulting electrochemically reduced product ions were analysed using TWIM-MS ( Figure S9 a to d respectively). It was found that only small contractions in CCS occurred as ions were reduced from 11+ to 5+ (Δ TW CCS N2 ca. 5 %, Figure  S10 and Table S1). By comparison to previously reported changes in CCS values associated with the electrochemical reduction induced molecular rearrangement of a foldamer over the same range of charge states (Δ CCS ca. 30%), [4] the small CCS changes observed here indicate that only subtle conformational changes occur during the reduction of the cages reported herein.

Electron Transfer Reaction Kinetics
Ion-ion reaction kinetics in an ion trap have been detailed by McLuckey and co-workers. [7] Primarily, the reaction rate is proportional to the square of the number of charges. [7b] As long as the local concentration of FL -• greatly exceeds that of the cation and q is held constant, the stepwise electrochemical reduction of multiply-charged cage cations can be described by pseudo-first order kinetics of consecutive reactions. [8] To confirm pseudo-first order conditions, rate constants were measured at different FL -• number densities by adjusting the automatic gain control. Above a critical AGC threshold (4 × 10 5 ), the obtained rate constants were found to be independent of the FL -• number density. [8b, 9] The reaction time was varied from 1 -200 ms in random order to account for temporal fluctuations in fluoranthene ion concentration.
The reaction of a cage ion in the z+ charge state (C z+ ) with FL -• forms a radical cation C (z−1)+• and neutral FL (Equation 1). Further reduction with a second FL -• yields C (z−2)+•• and so on. Rate constants were obtained using changes in the raw intensities of the precursor ion and each charge-reduced product ion as a function of reaction time. Schmidt and co-workers have described a method for estimating the rate constants of consecutive irreversible reactions. [10] First, k 1 was obtained by fitting the decay of C z+ to a single exponential equation (Equation 2). Next, the abundance of C (z−1)+ was plotted against reaction time and fitted to Equation 3 [10] with the least squares method using k 1 from Equation 2, yielding k 2 . The rate constants k 3 … k n were obtained sequentially in the same way, with each subsequent species fit using the rate constants calculated for the preceding reaction steps. The first term, ∑ (0) =1 was also refined during each step using the least squares method, correcting for differences in instrument mass sensitivity across the entire m/z range. Typically, the entire isotopic cluster would be integrated to calculate the ion abundance at each reaction time point, however when significant isobaric interference was present around the precursor ion as a consequence of imperfect ion isolation, as in the case for Ni-1 16+ (Figure S3), only a single, well-resolved isotope peak was used for all ions in the data set.
where ≠ for ≠ , C i is the abundance of the ith species and all undefined products are 1.
The reproducibility of rate constants determined using this method was assessed by comparing the results of a series of experiments repeated 7 times over a period of 10 days. Precursor ions for this study were selected based on which were obtained in the greatest abundance from the ESI ( Figure S6). The experiments involved the FL −• induced electrochemical reduction of precursor ions of Ni-1(OTf) 5 z+ (x = 2 to 11), or C 60 ⊂Ni-1(OTf) 5 y+ (y = 3 to 11) (Table S2). Using Equation 3, rate constants for each reduction step from each replicate were calculated and from these a mean rate constant for each reduction step was obtained. Percentage uncertainties in these mean rate constants were calculated using Equation 4 below, where k n(min) and k n(max) are the minimum and maximum rate constants across all replicates for reduction step n and k M(n) is the mean rate constant for reduction step n.
While all calculated uncertainties were found to be 5% or less, we conservatively associate an error of ± 10% on all reaction rate constants presented herein involving reactions of precursor ions of charge states 13 + to 2 + . The electrochemical reaction rate increases as the charge state of the precursor ion increases, and as a result, cages with higher charge states (14 + to 16 + ) react with fluoranthene radical anions at rates approaching the temporal resolution of the instrumentation available. Accordingly, we estimate the error in rate constants with these precursor ions to be as high as 20%.  Figure S19) and C 60 ⊂Ni-1(OTf) 5 11+ (Table S8, Figure S20). Experiments carried out over the period of 10 days. Absolute uncertainty in mean given within parentheses. To validate this method, further experimentation was carried out wherein each successive reduced species was re-isolated and reacted with FL −• . In this way, the reaction rate at each reduction step could be modelled using a single exponential equation. Rate constants obtained in this way (Table S3) were found to fall within experimental error of those calculated using Equation 3. Table S3: Comparison of rate constants (k n, as defined above, in s −1 (× 10 −3 )) for the 7-step sequential reduction of Ni-1(OTf) 7 9+ (Table S9, Figure S21) obtained using Equation 3 vs re-isolating each successive species for reaction with FL −• (i.e. k 1 is the rate constant for the reaction Ni-1(OTf) 7 9+ + FL −• → Ni-1(OTf) 7 8+• + FL). Absolute uncertainties provided in parentheses.

Cyclic Voltammetry
Cyclic voltammetry was performed on a 0.1 mM solution of cage Ni-1 with a Biologic SP-150 potentiostat under an argon atmosphere in anhydrous 1:4 acetonitrile:dimethyl formamide and 0.1M concentration of TBAPF 6 salt. All measurements were carried out in ca. 1 mL sample aliquots using a standard 3-electrode electrochemical cell. A glassy carbon electrode with a surface area of 0.07069 cm 2 was used as the main working electrode with rigorous cleaning in between each experiment. An aluminium fibre pad with a slurry of aluminium oxide polishing powder was used for gentle surface abrasion for surface particulates removal and subsequently washed with Milli-Q water and finally with acetone. A solid platinum wire counter electrode and an eDAQ leakless Ag/AgCl reference was used, rinsed with acetonitrile before use. Ferrocene was used as an external reference standard.
The cathodic sweep revealed a series of irreversible reduction processes indicating some degree of communication between the redox centres. Interestingly one quasi-reversible redox couple was observed in this range (−1.76 V vs. Fc/Fc + ) which we tentatively attribute to the single electron reduction/oxidation of the Ni porphyrin moiety based on related tetra-substituted nickel(II) porphyrins reduction potentials. [11] These results indicate that while a series of multiply reduced species akin to those observed in the gas phase can be formed in solution, investigations of the properties of the cage at each individual redox state are more accessible in the latter where purification through mass-selection is possible. Superscript numbers indicate the potential of observed redox peak. Oxidation peak at 0.57 V is believed to be oxidation of Fe(II) Fe(III) leading to irreversible collapse of the complex and passivation of the electrode surface.

Dioxygen Reactivity Investigations
Following reduction of an ion to the desired charge state with FL −• , the reduced ion was re-isolated using an isolation window of 15 Da and held in the ion trap with no supplemental collision energy for 200 to 2000 ms. After this time, the reaction products were scanned out and analysed in the Orbitrap.
Differences between observed isotopic distributions for O 2 reaction products ( Figure S15, S22) and the expected naturally occurring distributions are attributed to lower signal intensity, as well as the use of two subsequent mass selection processes (i.e., an MS 3 experiment), which may skew the observed isotopic pattern away from the naturally occurring distribution.  Table S4: Experimentally measured mass-to-charge ratios (m/z) of product ions (base peak of isotopic distribution) resulting from electrochemical reduction of cage Ni-1 with FL −• shown in Figure 3a. Figure S16: Comparisons of experimental isotopic distributions for selected electrochemically reduced ions of Ni-1 16+ (experimental top, theoretical bottom). Table S5: Experimentally measured mass-to-charge ratios (m/z) of product ions (base peak of isotopic distribution) resulting from electrochemical reduction of cage C 60 ⊂Ni-1 with FL −• shown in Figure S11.     2813.0486 0.8 Figure S20: Comparisons of experimental isotopic distributions for selected electrochemically reduced ions of C 60 ⊂Ni-1(OTf) 5 11+ (experimental top, theoretical bottom). 2672.3492 0.6 Figure S21: Comparisons of experimental isotopic distributions for selected electrochemically reduced ions of Ni-1(OTf) 7 9+ (experimental top, theoretical bottom).

Assignment
Table S10: Experimentally measured mass-to-charge ratios (m/z) of product ions (base peak of isotopic distribution) resulting from reaction of reduced cages with O 2 as shown in Figure 4. 2586.1319 3.5 Figure S22: Comparison of experimental isotopic distribution for ions of C 60 ⊂Ni-1 3+(13•) ·2O 2 (experimental top, theoretical bottom).